The initiation of bone remodeling depends in part on the support of osteoclast differentiation by stromal cells that appear to be of the osteoblast lineage. The key mechanism by which these stromal/osteoblastic cells support osteoclastogenesis is expression of RANKL (receptor activator of NFkappaB ligand). Hormones that stimulate osteoclast formation, such as PTH, do so by stimulating RANKL expression in stromal/osteoblastic cells but not other cell types. However, strikingly little is known about how this is accomplished. The long-term goal of this project is to identify the mechanisms that control RANKL expression in stromal/osteoblastic cells. Preliminary studies have determined that PTH stimulates RANKL transcription via a PKA-CREB pathway and that PTH also stimulates RANKL mRNA stability. Promoter-reporter constructs containing up to 7 kb of RANKL 5'-flanking region are regulated weakly or not at all by osteoclastogenic agents, suggesting that regulation of this gene involves distant regulatory elements. Consistent with this, a 160-kb DNA fragment harboring the murine RANKL gene as well as extensive flanking regions contains sequences sufficient for appropriate regulation by PKA in stably transfected stromal/osteoblastic cells. In Aim 1, cis-acting elements required for transcriptional control of RANKL expression by PTH will be identified by deletion analysis of the 160 kb DNA fragment and DNase I hypersensitivity assays. Elements will be further localized by DNase I protection and gel shift assays. Site-directed mutagenesis of the full-length construct will confirm the significance of each site. In Aim 2, cis-acting sequences that mediate post-transcriptional control of RANKL mRNA by PTH will be identified. Tagged murine RANKL mRNA will be conditionally expressed in stromal/osteoblastic cells and sequences involved in destabilization/PTH-induced stabilization will be mapped by deletion analysis. Aim 3 will identify regions of the RANKL gene involved in tissue-specific expression. This will be accomplished by creating transgenic mice harboring luciferase reporter constructs containing different portions of the RANKL gene and flanking region. Information gained from these studies will lead to a better understanding of the cellular factors involved in the support of osteoclast formation and thereby identify novel targets for anti-resorptive therapy.